Glycolysis is the metabolic pathway in eukaryotes that converts glucose into pyruvate. The free energy released in this process is used to form the high-energy compounds ATP (adenosine triphosphate), FADH2 and NADH (reduced nicotinamide adenine dinucleotide). Glycolysis is regulated by slowing down or speeding up certain steps in the glycolytic pathway, by inhibiting or activating the enzymes that are involved.
Phosphofructokinase is an important control point in the glycolytic pathway, since it is one of the irreversible steps and has key allosteric effectors. In the glycolysis pathway, phosphofructokinase-1 (PFK-1) catalyzes the major rate-limiting step that converts fructose-6-phosphate (Fru-6-P) to fructose-1,6-bisphosphate (Fru-1,6-BP). PFK-1 is allosterically regulated by fructose-2,6-bisphosphate (Fru-2,6-BP). Fru-2,6-BP is the most potent glycolysis stimulator.
Normally, under abundant energy supply, high levels of ATP strongly inhibit PFK-1 activity. However, Fru-2,6-BP can override this inhibitory effect and enhance glucose uptake and glycolytic flux. Of note, Fru-2,6-BP synthesis is up-regulated in many cancer cell lines.
Unlike normal cells, cancer cells have been noted to shift their energy metabolism toward glycolysis [34]. This phenomenon was originally termed the Warburg effect. This energy transition allows cancer cells to satisfy increased biosynthetic requirements for biomass and energy [35, 36]. Studies have consistently shown an abnormally high glycolytic rate in a wide spectrum of human cancers, but the causative mechanisms responsible for this metabolic adaptation remain poorly understood [37, 38]. Among the possible mechanisms, mitochondrial respiratory defects and hypoxia in the tumor microenvironment are attributed as two major factors for the Warburg effect [39, 40, 41].
Despite the complexity and obscurity of underlying mechanisms responsible for the Warburg effect, the metabolic consequences are a consistent transformation toward glycolysis as the major source of ATP production [37, 42]. This metabolic abnormality of cancer cells provides a potential biochemical basis to preferentially suppress progression of malignant cells by selective inhibition of glycolysis [43, 44, 45].
The upregulation of Fru-2,6-BP synthesis in many cancer cell lines has led to the suggestion that selective depletion of intracellular Fru-2,6-BP in cancer cells might be used to impede glycolytic flux, and thereby suppressing malignant cell survival and progression [49, 50, 51] A family of bifunctional enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFBs; a family of 4 isozymes, i.e., PFKFB isozymes 1-4), are responsible for the intracellular levels of Fru-2,6-BP [51, 52, 53]. Among these isozymes, PFKFB3 is dominantly over-expressed in thyroid, breast, colon, prostatic, and ovarian tumor cell lines [51, 54, 55]. PFKFB3 has kinase activity at least 10× that of the second most active isoform, and rapidly increases the level of Fructose-2,6-bisphosphate (F-2,6-BP).[15, 16] Recent studies have shown that induction of PFKFB3 expression by HIF-1 under hypoxic condition was followed by increased invasive potential and resistance to chemotherapies [54, 56].
Accordingly, an area of unmet need has been the ability to inhibit PFKFB3 as a therapeutic strategy for cancer [55]. Despite the potential merits, exploitation of the inhibition of PFKFB3 for cancer therapy has remained an unmet need. A pyridinyl-containing compound has been reported as a possible PFKFB3 inhibitor, based on the receptor structure predicted from that of PFKFB4 [57; see also, U.S. Published Patent Application No. 2009/0074884]. Although promising, inhibitors based on structures other than the potent PFKFB3 enzyme may lack specificity and limit desired improvement of inhibitor potency.